In vitro Metabolism of Halofantrine

Mouse, rat, dog and human intestinal microsomes and mouse, rat and human liver microsomes were obtained from Xenotech, Lenexa, KS. Dog liver microsomes were obtained from BD Gentest, Discovery Labware, Inc. Metabolic stability of halofantrine was assessed in vitro by incubating at 37°C with microsomes suspended in 0.1 M phosphate buffer (pH 7.4) at a final halofantrine concentration of 0.5 μM and microsomal protein concentration of 0.4 mg/mL (Doggett et al., 2012; Carosati et al., 2016).

Metabolic reactions were initiated by the addition of a NADPH-regenerating system (1 mg/mL NADP, 1 mg/mL glucose-6-phosphate, and 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl₂ (0.67 mg/mL) and were quenched by the addition of ice-cold acetonitrile at five time points (2, 5, 15, 30, and 60 min) (Doggett et al., 2012). Compounds were also incubated in the absence of NADPH to monitor for cofactor independent degradation in the microsomal matrix (2, 30, and 60 min). No compound loss was observed in the absence of NADPH. Quenched samples were centrifuged, and the clear supernatant was analyzed to monitor the extent of drug loss using the LC–MS assay described below. Concentration versus time data for each compound were fitted to an exponential decay function to determine the first order rate constant for substrate depletion, which was then used to calculate the degradation half-life and an in vitro intrinsic clearance (Cl_int, in vitro) value (mL/min/mg microsomal protein).

A cocktail of known compounds (dextromethorphan, diclofenac, midazolam) was included in the incubation alongside test compounds, and the degradation half-life (min) values observed were in agreement with historical values, thereby validating the assay conditions employed (Obach, 1999; Doggett et al., 2012; Carosati et al., 2016).

Halofantrine concentrations in samples from the microsome metabolism studies were assayed using the same HPLC–MS assay as used for the mouse studies as described and validated previously (Trevaskis et al., 2013).